--- - |2+ Abstract Aim Endothelial glycocalyx (EG) plays a pivotal role in a plethora of diseases, like cardiovascular and renal diseases. One hallmark function of the EG as a mechanotransducer which transmits mechanical signals into cytoplasm has been documented for decades. However, the basic question ‐ how the glycocalyx transmits the flow shear stress— is unanswered so far. Our aim is to shed light on the fundamental mode of signal transmission from flow to the endothelial cytoskeleton. Methods We conduct a series of large‐scale molecular dynamics computational experiments to investigate the dynamics of glycocalyx under varying conditions (changing blood flow velocities and shedding of glycocalyx sugar chains). Results We have identified that the main pathway of signal transmission in this system manifests as a scissors‐like motion of the Syndecan‐4 core protein. Results have suggested that the force transmitted into the cytoskeleton with an order of 10 ~ 100 pN, and the main function of sugar chains of a glycocalyx element is to protect the core proteins from severe conformational changes thereby maintaining the functionality of the EG. Conclusion This research provides a reconciling explanation for a longstanding debate about the force transmission threshold based on our findings. A new explanation has also been provided to relate the role of the EG as a mechanotransducer to its function as a microvascular barrier: the EG regulates the mechanotransduction by altering the median value and variation range of the scissor angle, and the EG governs the microvascular barrier via controlling the scissor angle which will affect the intercellular cleft. - 'Acta Physiologica, EarlyView. '